NANOPARTICLE COMPOSITION AND ASSOCIATED
METHODS THEREOF
BACKGROUND
[0001] This invention relates generally to nanoparticle compositions which form
stable aqueous suspensions, particularly nanoparticle compositions based on transition
metal oxides. Such nanoparticle compositions are useful for a variety of applications
including diagnostic imaging.
[0002] Nanoparticles, i.e. particles whose diameters are appropriately measured in
nanometers, have been considered for a wide variety of end uses. Some of the uses
require a substantial degree of hydrophilicity. However, in a number of instances, the
material upon which nanoparticles are based may lack this attribute. For instance,
nanoparticles with appropriate imaging properties for use as contrast agents for MR
and/or X-ray imaging are typically based on transition metal oxides which lack the
level of hydrophilicity required to form the stable aqueous suspensions needed for
such applications. Therefore, efforts have been made to modify the surface properties
of such nanoparticles to be more compatible with aqueous media and thereby enhance
the stability of aqueous suspensions of such nanoparticles. In some applications, it is
also desirable that the nanoparticles have a relatively monodisperse particle size
distribution. However, such surface treatments typically result in a relatively
polydisperse particle size distribution.
[0003] Typically, nanoparticle compositions in aqueous suspension are subject to
agglomeration and precipitation of the constituent nanoparticles. Surface treatments
may be used to inhibit such agglomeration and precipitation, and may take the form of
adding one or more stabilizer substances to a suspension of a nanoparticulate core
species in a diluent. Such stabilizer substances are thought to attach to the surface of
the suspended nanoparticulate core species and to form a barrier (or shell) interposed
between at least a portion of the surface of the nanoparticulate core species and the
diluent in which the nanoparticulate core species are suspended.[0004] Formulations comprising nanoparticle compositions suitable for use in
medical imaging applications typically require purification prior to presentation to a
subject. The various purification techniques employed may degrade the
hydrophilicity of the nanoparticle composition and may alter the particle size
distribution of the nanoparticle composition. Prudent medical practice and logic
strongly suggest that formulations containing nanoparticle compositions to be used as
contrast agents for in vivo use in human subjects will be subjected to rigorous
purification and be required to exhibit robust suspension stability in isotonic aqueous
media, for example in 150 mM sodium chloride solution.
[0005] Thus, there is a need for nanoparticle compositions with improved properties,
particularly related to increased hydrophilicity, stability in colloidal suspension, and
enhanced safety.
BRIEF DESCRIPTION
[0006] In one embodiment the present invention provides a nanoparticle composition
comprising a nanoparticulate metal oxide; and a phosphorylated polyol comprising at
least two phosphate groups, wherein the polyol comprises one or more hydrophilic
groups selected from the group consisting of polyethylene ether moieties,
polypropylene ether moieties, polybutylene ether moieties, and combinations of two
or more of the foregoing hydrophilic moieties. In a further embodiment the present
invention provides a diagnostic agent composition suitable for injection into a
mammalian subject comprising such nanoparticle composition.
[0007] In another embodiment, the present invention provides a nanoparticle
composition comprising a nanoparticulate iron oxide core; and a shell comprising a
phosphorylated polyol comprising at least two phosphate groups, wherein at least two
of the phosphate groups occupy positions in the phosphorylated polyol which
constitute a 1,2 or 1,3 spatial relationship to one another and the polyol comprises a
hydrophilic group selected from the group consisting of polyethylene ether moieties,
polypropylene ether moieties, polybutylene ether moieties, and combinations of two
or more of the foregoing hydrophilic moieties. In a further embodiment, the presentinvention provides a diagnostic agent composition suitable for injection into a
mammalian subject comprising such nanoparticle composition.
[0008] In yet another embodiment, the present invention provides a nanoparticle
composition comprising a nanoparticulate metal oxide core, wherein the metal oxide
comprises a metal selected from the group consisting of iron, tantalum, zirconium,
and hafnium; and a shell comprising a phosphorylated polyol comprising at least two
phosphate groups, wherein at least two of the phosphate groups occupy positions in
the phosphorylated polyol which constitute a 1,2 or 1,3 spatial relationship to one
another and the polyol comprises a hydrophilic group selected from the group
consisting of polyethylene ether moieties, polypropylene ether moieties, polybutylene
ether moieties, and combinations of two or more of the foregoing hydrophilic
moieties. In yet a further embodiment, the present invention provides a diagnostic
agent composition suitable for injection into a mammalian subject, comprising such
nanoparticle composition.
[0009] In yet another embodiment, the present invention provides a process for
making a nanoparticle composition comprising contacting a nanoparticulate metal
oxide core with a shell composition comprising a phosphorylated polyol comprising
at least two phosphate groups and one or more hydrophilic groups selected from the
group consisting of polyethylene ether moieties, polypropylene ether moieties,
polybutylene ether moieties, and combinations of two or more of the foregoing
hydrophilic moieties.
[0010] In yet another embodiment, the present invention provides a process of
diagnostic imaging comprising: (a) administering a diagnostic agent composition to a
subject, wherein the diagnostic agent composition comprises a nanoparticle
composition comprising a nanoparticulate metal oxide selected from the group
consisting of iron oxide, manganese oxide, tantalum oxide, zirconium oxide, hafnium
oxide, and combinations of two or more of the foregoing metal oxides; and a
phosphorylated polyol comprising at least two phosphate groups and one or more
hydrophilic groups selected from the group consisting of polyethylene ether moieties,
polypropylene ether moieties, polybutylene ether moieties, and combinations of twoor more of the foregoing hydrophilic moieties; and a pharmaceutically acceptable
carrier or excipient; and (b) subjecting the subject to diagnostic imaging, wherein the
nanoparticle composition acts as a contrast agent.
BRIEF DESCRIPTION OF THE FIGURES
[0011] These and other features, aspects, and advantages of the present invention will
become better understood when the following detailed description is read with
reference to the accompanying drawings in which like characters represent like parts
throughout the drawings, wherein:
[0012] FIG. 1 is an idealized cross sectional view of a nanoparticle comprising a core
and a shell, in accordance with one embodiment of the present invention.
[0013] FIG. 2A is a Ti weighted image (TE = 4.1 ms) of a tumor in accordance with
Example 29, before administration of iron oxide nanoparticle composition.
[0014] FIG. 2B is a Ti weighted image (TE = 4.1 ms) of a tumor in accordance with
Example 29, 30 min after the administration of the nanoparticle contrast agent of
Example 10.
[0015] FIG. 2C is a difference map of the differences between FIG. 2A and FIG. 2B.
[0016] FIG. 2D is a T2*-weighted image (TE = 24.5 ms) of a tumor in accordance
with Example 29, before administration of iron oxide nanoparticle composition.
[0017] FIG. 2E is a T2*-weighted image (TE = 24.5 ms) of a tumor in accordance
with Example 29, 15 min after the administration of the nanoparticle contrast agent of
Example 10.
[0018] FIG. 2F is an R2* relaxation difference map of the differences between FIG.
2D and FIG. 2E exhibiting a clear distinction between tumor and muscle tissue.
DETAILED DESCRIPTION[0019] In the following specification and the claims which follow, reference will be
made to a number of terms, which shall be defined to have the following meanings.
[0020] The singular forms "a", "an" and "the" include plural referents unless the
context clearly dictates otherwise.
[0021] "Optional" or "optionally" means that the subsequently described event or
circumstance may or may not occur, and that the description includes instances where
the event occurs and instances where it does not.
[0022] As used herein, the term "solvent" can refer to a single solvent or a mixture of
solvents.
[0023] Approximating language, as used herein throughout the specification and
claims, may be applied to modify any quantitative representation that could
permissibly vary without resulting in a change in the basic function to which it is
related. Accordingly, a value modified by a term or terms, such as "about", is not to
be limited to the precise value specified. In some instances, the approximating
language may correspond to the precision of an instrument for measuring the value.
[0024] Unless specified otherwise, as used herein the term "phosphate group" refers
to the bracketed group I shown below (and its ionized forms II and III) and includes
four constituent oxygen atoms and one constituent phosphorous atom but does not
include the carbon atom shown. The phosphate group is linked through one of its four
oxygen atoms via a bond (see dashed line) to a carbon atom in an organic moiety, the
phosphate group and the organic moiety forming constituents of an organic molecule,
for example a phosphorylated polyol (See illustrative examples in the Experimental
Section of this disclosure). Because phosphate groups readily ionize to the
corresponding mono anionic (See group II) and dianionic (See group III) forms, the
term phosphate group as used herein includes each of these forms in addition to the
fully protonated form featured in group I . The relative amounts of each of the forms
I-III of a phosphate group present in, for example, a phosphorylated polyol, will
depend on the environment in which the phosphate group is present. At high pH in
aqueous media there should be more of form III relative to form I, for example. Inaddition, for the purposes of his disclosure, the term
phosphate group specifically excludes "polyphosphates" in which a first phosphorous
atom is linked to a second phosphorous atom via an oxygen atom without an
intervening carbon atom. Structure IV below illustrates a polyphosphate as defined
herein. As defined herein, a polyphosphate comprises a first phosphorous atom (P1)
linked to a second phosphorous atom (P2) via an oxygen atom without an intervening
carbon atom. In the polyphosphate illustrated in structure IV the
IV
polyphosphate group comprises seven oxygen atoms and two phosphorous atoms. An
alternate illustrative polyphosphate group includes ten oxygen atoms and three
phosphorous atoms. As illustrated in structure IV a polyphosphate group is linked to
a moiety Q which may be an organic moiety or an inorganic moiety. Polyphosphoric
acid illustrates an example of a polyphosphate in which Q is an inorganic moiety.
Trisodium O-methyl diphosphate illustrates an organic diphosphate wherein Q is a
methyl group and the OH groups attached to phosphorous are ionized and attended by
charge-balancing counterions (three sodium cations) (Chemical Papers 62 (2) 223-226
(2008)). Those of ordinary skill in the art will appreciate that as defined herein, the
term polyphosphate encompasses both "acyclic polyphosphates" (wherein neither of
the first phosphorous atom (P1) linked to the second phosphorous atom (P2) via an
oxygen atom without an intervening carbon atom is part of a cyclic structure) and
"cyclic polyphosphates" (wherein in which at least one of the first phosphorous atom
(P1) linked to the second phosphorous atom (P2) via an oxygen atom without an
intervening carbon atom is part of a cyclic structure). Those of ordinary skill in theart will further appreciate that there are various ionized forms of polyphosphates and
that the term polyphosphate is meant to include the ionized forms of an idealized fully
protonated polyphosphate, for example the fully protonated polyphosphate structure
shown in structure IV above.
[0025] As discussed in detail below, embodiments of the present invention include a
nanoparticle composition comprising a nanoparticulate metal oxide, and a
phosphorylated polyol, wherein the phosphorylated polyol comprises at least two
phosphate groups and a hydrophilic group, wherein the phosphate groups are
chemically and sterically accessible to the metal oxide and the hydrophilic group is
selected from the group consisting of polyethylene ether moieties, polypropylene
ether moieties, polybutylene ether moieties, and combinations of two or more of the
foregoing hydrophilic moieties.
[0026] In various embodiments, the nanoparticle compositions provided by the
present invention are sufficiently hydrophilic to form stable aqueous colloidal
suspensions that exhibit no substantial change in the hydrodynamic diameter (DH) of
constituent nanoparticles over a prolonged time frame (e.g. over several days to
several weeks). A change in hydrodynamic diameter over time is a key indicator of
colloidal suspension stability. Thus, nanoparticle compositions that display robust
stability in colloidal suspension should show little or no increase in the hydrodynamic
diameter (DH) of the suspended constituent nanoparticles over the time period of
interest. Hydrodynamic diameter may be measured by dynamic light scattering
(DLS). Those of ordinary skill in the art will understand that the term hydrodynamic
diameter (DH) refers to the average hydrodynamic diameter.
[0027] As used herein, the term 'nanoparticle composition' refers to a composition
comprising constituent nanoparticles having average particle size of less than 1
micrometer. As used herein, the term 'size' refers to the hydrodynamic diameter of
the nanoparticles. In one embodiment, the nanoparticle composition provided by the
present invention has a DH in a of range from about 2 nm to about 500 nm. In an
alternate embodiment, the nanoparticle composition provided by the present invention
has a DH in a range of from about 10 nm to 25 nm. In one embodiment, thenanoparticle composition provided by the present invention has a DH of less than 50
nm. In another embodiment, the nanoparticle composition provided by the present
invention has a DH of less than 10 nm. In yet another embodiment, the nanoparticle
composition provided by the present invention has a DH of less than 5 nm. A small
particle size may be advantageous in, for example, facilitating clearance of the
nanoparticle composition from the kidneys and other organs of a subject following a
medical imaging procedure employing the nanoparticle composition as a contrast
agent.
[0028] In one embodiment, the nanoparticle composition provided by the present
invention comprises a core-shell structure, wherein the core comprises a
nanoparticulate metal oxide, and the shell comprises a phosphorylated polyol
comprising at least two phosphate groups and one or more hydrophilic groups
selected from the group consisting of polyethylene ether moieties, polypropylene
ether moieties, polybutylene ether moieties, and combinations of two or more of the
foregoing hydrophilic moieties.
[0029] In various embodiments, the shell comprising the phosphorylated polyol
stabilizes the nanoparticulate metal oxide core and prevents the formation of larger
metal oxide particles by association (agglomeration) of the nanoparticulate metal
oxide core particles. One or more embodiments of the invention are related to a
nanoparticle composition having the idealized core-shell structure shown in FIG. 1.
The nanoparticle composition 10 comprises a nanoparticulate metal oxide core 12,
and a shell 14 comprising a phosphorylated polyol as described herein. In one
embodiment, the present invention provides a nanoparticle composition characterized
by its ability to form a stable aqueous colloidal suspension that exhibits no substantial
change in hydrodynamic diameter (DH) as determined by dynamic light scattering in
150 mM aqueous NaCl after tangential flow filtration and storage for one week at
room temperature.
[0030] The metal oxide core of the nanoparticle composition provided by the present
invention has dimensions appropriately measured in nanometers. In various
embodiments, the nanoparticulate metal oxide core may be prepared as a suspensionin a diluent and the hydrodynamic diameter of the suspended nanoparticulate metal
oxide core particles may be measured, for example by dynamic light scattering. In
one embodiment, the nanoparticulate metal oxide core has a DH as measured by
dynamic light scattering in a range from about lnm to about 30 nm. In an alternate
embodiment, the nanoparticulate metal oxide core has a DH as measured by dynamic
light scattering of about 5 nm. In one or more embodiments, the nanoparticulate
metal oxide core comprises a nanoparticulate super paramagnetic iron oxide (SPIO)
and has a DH as measured by dynamic light scattering of less than about 25 nm.
[0031] The nanoparticulate metal oxide core typically comprises a transition metal
oxide. In one embodiment, the nanoparticulate metal oxide core consists of a single
transition metal oxide, for example tantalum oxide alone or iron oxide alone. In
another embodiment, the nanoparticulate metal oxide core comprises two or more
transition metal oxides. Thus in one embodiment the nanoparticulate metal oxide
core comprises both tantalum oxide and hafnium oxide. In various embodiments, the
nanoparticulate metal oxide core may comprise additional materials not constituting
metal oxides, such as metal nitrides and metal sulfides. Thus, in one embodiment the
nanoparticulate metal oxide comprises tantalum oxide and hafnium nitride. In yet
another embodiment, the nanoparticulate metal oxide core comprises tantalum oxide
and tantalum sulfide.
[0032] In one embodiment, the nanoparticulate metal oxide core comprises a
transition metal oxide selected from the group consisting of oxides of tungsten,
tantalum, hafnium, zirconium, zinc, molybdenum, silver, iron, manganese, copper,
cobalt, nickel and combinations of two or more of the foregoing transition metal
oxides. In one specific embodiment, the transition metal oxide is tantalum oxide. In
an alternate embodiment, the transition metal oxide is iron oxide. Typically, the
nanoparticulate metal oxide core comprises at least 30% by weight of the transition
metal component of the transition metal oxide. In one embodiment, the
nanoparticulate metal oxide core comprises at least 50% by weight of the transition
metal component. In yet another embodiment, the nanoparticulate metal oxide core
comprises at least 75% by weight of the transition metal component. Those of
ordinary skill in the art will appreciate that a relatively high transition metal content inthe nanoparticulate metal oxide core can provide nanoparticle compositions with a
relatively higher degree of radiopacity per unit volume, thereby imparting more
efficient performance as a contrast agent.
[0033] For use as X-ray contrast agents, the nanoparticle composition provided by the
present invention should be substantially more radiopaque than the tissue and bone
matter typically found in living organisms. In certain embodiments, the present
invention provides nanoparticle compositions comprising nanoparticulate metal oxide
cores comprising metal atoms having an atomic number greater than or equal to 34.
Such nanoparticle compositions may be effective as imaging agents when presented
to a subject in a medical imaging formulation having a nanoparticle composition
concentration sufficient to provide an effective metal concentration in the subject's
blood during the imaging procedure of approximately 50 mM. Such materials are
likely yield appropriate contrast enhancement of about 30 Hounsfield units (HU) or
greater. Of special interest are materials that lead to a contrast enhancement in a
range from about 100 Hounsfield to about 5000 Hounsfield units. Examples of
transition metal elements that may provide this property include tungsten, tantalum,
hafnium, zirconium, molybdenum, silver, and zinc. In one embodiment, the present
invention provides a nanoparticle composition suitable for use in X-ray imaging
applications such as computed tomography (CT), the nanoparticle composition
comprising a nanoparticulate metal oxide core comprising tantalum oxide.
[0034] In one or more embodiments, the core of the nanoparticle composition
comprises tantalum oxide with a particle size up to about 6 nm. Such embodiments
may be particularly attractive in imaging techniques that apply X-rays to generate
imaging data, due to the high degree of radiopacity of the tantalum-containing core
and the small size that aids rapid renal clearance, for example.
[0035] In some embodiments, the metal oxide core comprises a transition metal,
which exhibits magnetic behavior, including, for example, superparamagnetic
behavior. In some embodiments, the metal oxide core comprises a paramagnetic
metal, selected from the group consisting of iron, manganese, copper, cobalt, nickel,
and combinations thereof. In a specific embodiment, the metal oxide core comprisessuperparamagnetic iron oxide (SPIO). In one embodiment, the iron oxide is doped
with another metal.
[0036] In some embodiments, the nanoparticle compositions of the present invention
may be used as magnetic resonance (MR) contrast agents. For use as MR contrast
agents the nanoparticle composition provided by the present invention advantageously
comprises a paramagnetic metal species, with those compositions that comprise a
superparamagnetic metal species being of particular interest. Examples of potential
paramagnetic and superparamagnetic materials include materials comprising one or
more of iron, manganese, copper, cobalt, nickel or zinc. A particularly interesting
group of materials are those based upon iron oxide, especially SPIO's, which typically
comprise from about 65% to about 75% iron by weight. In one embodiment, the
nanoparticulate metal oxide core comprises a iron compound having general formula
[Fe2
+03]x[Fe2
+03(M2+0)]i-
x wherein 1 > x > 0 and M2+ is a metal cation such as
cations of iron, manganese, nickel, cobalt, magnesium, copper, zinc and a
combination of such cations. Examples of iron compounds falling within the scope of
this general formula include magnetite (Fe3C"4) when the metal cation (M2+) is ferrous
ion (Fe2+) and x = 0; and maghemite (y-Fe20 3) when x = 1.
[0037] As shown in the idealized structure shown in FIG. 1, the nanoparticle
composition may comprise a shell which completely covers the nanoparticulate metal
oxide core. Thus, in certain embodiments, the nanoparticle composition is said to
comprise a shell which substantially covers the core. As used herein, the term
"substantially covers" means that a percentage surface coverage of the core by the
shell is greater than about 20%. As used herein, the term percentage surface coverage
refers to the ratio of the core surface covered by the shell to the core surface not
covered by the shell. In some embodiments, the percentage surface coverage of the
nanoparticle may be greater than about 40%.
[0038] In some embodiments, the shell may facilitate improved water solubility,
reduce aggregate formation, prevent oxidation of nanoparticles, maintain the
uniformity of the core-shell entity, and/or provide biocompatibility for the
nanoparticle compositions.[0039] The average thickness of shell is typically in a range from about 1 to about
50nm. In one embodiment, the shell has an average thickness less than 50 nm. In
another embodiment, the shell has an average thickness of less than 8 nm. In yet
another embodiment, the shell has an average thickness of less than 5 nm.
[0040] The nanoparticle compositions provided by the present invention may
comprise more than one shell layer disposed on the nanoparticulate metal oxide core.
By judicious selection of processing conditions, a nanoparticulate metal oxide core
species may be prepared as a suspension in a diluent and thereafter treated under a
first set of conditions with one or more stabilizer substances to generate a first
nanoparticle composition comprising a first shell, and thereafter the first nanoparticle
composition is treated under a second set of conditions with one or more different
stabilizer substances which generate a second nanoparticle composition comprising
both the first shell and a second shell. In embodiments comprising a plurality of
shells, at least one of the shells comprises a phosphorylated polyol comprising at least
two phosphate groups and one or more hydrophilic groups selected from the group
consisting of polyethylene ether moieties, polypropylene ether moieties, polybutylene
ether moieties, and combinations of two or more of the foregoing hydrophilic
moieties. In one embodiment, a single shell may cover essentially the entire surface
of the nanoparticulate metal oxide core. In another embodiment, the present invention
provides a nanoparticle composition comprising a single nanoparticulate metal oxide
core composition and multiple shell compositions, as in the case where a
nanoparticulate metal oxide core species is prepared as a suspension in a diluent, the
suspension is divided in half and each half is treated with a different phosphorylated
polyol, and subsequently the halves are recombined. Thus, within a nanoparticle
composition provided by the present invention, individual particles may comprise
shells which are essentially identical to the shells of companion particles within the
nanoparticle composition; or the shells of constituent particles within the nanoparticle
composition may differ from one another in composition.
[0041] As noted, the nanoparticle compositions provided by the present invention
comprise a phosphorylated polyol, the phosphorylated polyol comprising at least two
phosphate groups and one or more hydrophilic groups. The hydrophilic group (orgroups) is selected from the group consisting of polyethylene ether moieties,
polypropylene ether moieties, polybutylene ether moieties, and combinations of two
or more of the foregoing hydrophilic moieties. Polyethylene ether moieties are
defined as moieties comprising oxyethyleneoxy structural units -OCH2CH20-, and/or
substituted oxyethyleneoxy structural units. For convenience and because of the close
structural association with the term polyethylene glycol (PEG), such moieties may at
times herein be referred to as PEG groups, or PEG moieties, and are characterized by
a moiety molecular weight. Illustrative polyethylene ether moieties are given in Table
1 below and throughout this disclosure. Similarly, polypropylene ether moieties are
defined as moieties comprising oxypropyleneoxy structural units -OCH2CH2CH20 -
and/or substituted oxypropyleneoxy structural units. For convenience polypropylene
ether moieties may at times herein be referred to as polypropylene glycol groups or
moieties. Similarly, polybutylene ether moieties are defined as moieties comprising
oxybutyleneoxy structural units -OCH2CH2CH2CH20 - and/or substituted
oxybutyleneoxy structural units. For convenience polybuylene ether moieties may at
times herein be referred to as poly-TFIF moieties.
[0042] Illustrative phosphorylated polyols used in, and provided by the present
invention are given in Table 1 below. In each of Entries la- If, the illustrated
phosphorylated polyol comprises at least two phosphate groups and one or more
hydrophilic groups selected from the group consisting of one or more of a
polyethylene ether moieties, polypropylene ether moieties, polybutylene ether
moieties, and combinations of two or more of the foregoing hydrophilic moieties.Table 1Exemplary Phosphorylated Polyols and Constituent Structural Elements[0043] As will be appreciated by those of ordinary skill in the art the phosphate
groups present in the phosphorylated polyol may be configured such that two
phosphate groups within the same phosphorylated polyol occupy positions which
constitute a 1,2; 1,3; 1,4; 1,5; or 1,6 spatial relationship to one another. In Table 1
Example l a illustrates a phosphorylated polyol in which two phosphate groups areconfigured in a 1,3 spatial relationship with respect to each other. Example lb
illustrates a phosphorylated polyol in which two phosphate groups are configured in a
1.2 spatial relationship with respect to each other. Those of ordinary skill in the art
will be familiar with such distinctions. A 1,2 spatial relationship of the at least two
phosphate groups includes embodiments which are 1,2-bisphosphates; 2,3-
bisphosphates; 3,4-bisphosphates; 4,5- bisphosphates, 5,6-bisphosphates and so on. A
1.3 spatial relationship of the at least two phosphate groups includes embodiments
which are 1,3 -bisphosphates; 2,4-bisphosphates; 3,5-bisphosphates; 4,6-
bisphosphates; 5,7- bisphosphates and so on. Those of ordinary skill in the art will
fully understand the extension of this principle to 1,4; 1,5; and 1,6 spatial
relationships of the at least two phosphate groups.
[0044] As noted, the phosphorylated polyol comprises one or more hydrophilic
groups selected from the group consisting of polyethylene ether moieties,
polypropylene ether moieties, polybutylene ether moieties, and combinations of two
or more of the foregoing hydrophilic moieties. The effectiveness of the
phosphorylated polyol in stabilizing the nanoparticulate metal oxide core (and the
nanoparticle composition as a whole) has been found to depend upon its structure. In
various embodiments, the effectiveness of the phosphorylated polyol in stabilizing the
nanoparticulate metal oxide core is dependent upon the size of the hydrophilic moiety
which may at times herein be described in terms of the group molecular weight of the
hydrophilic group. In general, the structure of the phosphorylated polyol may be
tailored to be effective in stabilizing a particular nanoparticulate metal oxide core, and
the hydrophilic group present in the phosphorylated polyol may have either a
relatively low group molecular weight (e.g. less than 100 grams per "mole") or a
relatively high group molecular weight (e.g. more than 10,000 grams per "mole").
Those of ordinary skill in the art will understand that because the hydrophilic group
comprises one or more of a polyethylene ether moiety, a polypropylene ether moiety,
a polybutylene ether moiety, and combinations of two or more of the foregoing
hydrophilic moieties, the size and molecular weights of these moieties, at times herein
referred to as moiety molecular weight, will contribute to the group molecular weight
of the hydrophilic group as a whole. In one embodiment, the hydrophilic groupcomprises a polyethylene ether moiety having a moiety molecular weight in a range
from about 750 daltons to about 20,000 daltons. In an alternate embodiment, the
hydrophilic group comprises a polyethylene ether moiety having a moiety molecular
weight of about 2000 daltons. In yet another embodiment, the hydrophilic group
comprises a polyethylene ether moiety having a moiety molecular weight of less than
20,000 daltons. In yet still another embodiment, the hydrophilic group comprises a
polyethylene ether moiety having a moiety molecular weight of less than 2000
daltons. In yet another embodiment, the hydrophilic group comprises a polyethylene
ether moiety having a moiety molecular weight of less than 350 daltons. As used
herein, "daltons" and "grams per mole" may be used as interchangeable terms which
when applied either to the group molecular weight of a hydrophilic group or the
moiety molecular weight of a polyethylene ether moiety, polypropylene ether moiety,
polybutylene ether moiety, combinations of two or more of the foregoing hydrophilic
moieties, and substituted variants of such moieties, and expresses the weight in grams
of the that group or moiety present in a mole of the phosphorylated polyol which
contains it.
[0045] The intended end use of the nanoparticle composition may impact the
selection of the hydrophilic groups used in the phosphorylated polyol. For instance,
where the nanoparticle compositions are to be used in vivo, particularly in human
subjects, it may be desirable to avoid hydrophilic groups containing ionic groups
which might bind strongly to tissue components such as proteins. For in vivo use,
hydrophilic groups with essentially no net charge, such as polyalkylene ethers are of
particular interest. In addition, for use in human subjects, hydrophilic groups that are
innocuous and permit the nanoparticle composition to be easily and reproducibly
characterized for safety evaluation are particularly desirable. The nanoparticle
composition provided by the present invention typically has a zeta potential in a range
from about -40 mV and +40 mV.
[0046] In one embodiment, the phosphorylated polyol has structure Vwherein n is an integer from about 6 to about 150 and R1 is an alkyl group or a
hydrogen atom. The phosphorylated 1,2-diol V is illustrated by phosphorylated
polyol 10 (Experimental Section Example 5, n = 10, R1 = methyl) also referred to
herein as 1,2BPP440. Phosphorylated 1,2-diol V is further illustrated by
phosphorylated polyol 15 (Experimental Section Example 7, n = 17, R1= methyl) also
referred to herein as 1,2BPP750. In one embodiment, the present invention provides
a phosphorylated 1, 2-diol having structure V wherein n is in a range from about 16 to
about 150 and R1 is an alkyl group or a hydrogen atom. See, for example,
phosphorylated 1, 2-diol 20 ((Experimental Section Example 9, n = 44, R1= methyl).
[0047] In an alternate embodiment, the phosphorylated polyol has structure VI
wherein n is an integer from about 6 to about 150 and R1 is an alkyl group or a
hydrogen atom. The phosphorylated 1,3-diol VI is illustrated by phosphorylated
polyol 27 (Experimental Section Example 13, n = 7, R1 = methyl) also referred to
herein as 1,3BPP350. In one embodiment, the present invention provides a
phosphorylated 1, 3-diol having structure VI wherein n is in a range from about 16 to
about 150 and R1 is an alkyl group or a hydrogen atom. See, for example,phosphorylated 1, 3-diol 3 1 ((Experimental Section Example 15, n = 44, R1= methyl)
also referred to herein as 1,3BPP2000.
[0048] In yet another embodiment, the phosphorylated polyol comprising at least two
phosphate groups and one or more hydrophilic groups has structure XVIII
XVI I I
wherein O-R2 is independently at each occurrence a phosphate group, a hydroxy
group, or a polyethylene ether moiety.
[0049] As used herein in relation to phosphorylated polyols and nanoparticle
compositions comprising such phosphorylated polyols or nanoparticle compositions
comprising structural units derived from such phosphorylated polyols, the designation
"1,2-BPP350" refers to a phosphorylated polyol comprising two phosphate groups
configured in a 1,2 spatial relationship and a polyethylene ether moiety having a
moiety molecular weight of 350 daltons. Similarly, the designation "1,2-BPP440"
refers to a phosphorylated polyol comprising two phosphate groups configured in a
1,2 spatial relationship and a polyethylene ether moiety having moiety molecular
weight of 440 daltons.
[0050] As used herein the designation P2P4Man refers to a phosphorylated mannitol
comprising approximately two phosphate groups per mannitol residue and
approximately four hydrophilic groups comprising polyethylene ether moieties.
Structure 23 in the Experimental Section illustrates such a mannitol-based
phosphorylated polyol.
[0051] Nanoparticle compositions provided by the present invention are illustrated by
structures VII-XVI below wherein the disc-shaped component labeled Fe3C"4
represents a nanoparticulate metal oxide core and the associated phosphorylatedpolyol structure represents one or more phosphorylated polyols bound to the
nanoparticulate metal oxide core. Structures VII-XVI are not meant to suggest a 1:1
stoichiometry between the nanoparticulate metal oxide core and the phosphorylated
polyol, but rather to identify the nanoparticle composition as comprising a the
nanoparticulate metal oxide care and at least one phosphorylated polyol. As noted,
the phosphorylated polyol may be in a fully protonated form as shown in structures
VII-XVI, or in an ionized form. (See Forms II and III herein). Typically, a plurality
of phosphorylated polyols will be associated with the surface of a given
nanoparticulate metal oxide core particle. In some embodiments, the phosphorylated
polyol is bound to the nanoparticulate metal oxide core via hydrogen bonds. In some
embodiments, the phosphorylated polyol is bound to the nanoparticulate metal oxide
core via at least one covalent bond. In other embodiments, the phosphorylated polyol
may be bound to the nanoparticulate metal oxide core via ionic bonds. In certain
embodiments, the precise nature of the chemistry through which the phosphorylated
polyol is bound to the nanoparticulate metal oxide core may not be well understood.
Notwithstanding such uncertainty, basic structure-activity principles for a variety of
such nanoparticle compositions provided by the present invention may be discerned
through experimentation, and such experimentally determined structure-activity
principles are disclosed herein.1.2BPP2000Ester[0052] As illustrated in structures XI, XII, XIII and XIV the phosphorylated polyol
component of the nanoparticle composition may, in certain embodiments, comprise a
hydrophilic group containing groups in addition to the ether linkages (-0-) found in
polyalkylene ether moieties. Thus, a wide variety of functional groups in addition to
ether groups may be present in the phosphorylated polyol, for example ester groups,
amine groups, amide groups, carbamate groups, urea groups, carbonate groups,
thioether groups, selenoether groups, siloxane groups, sulfinyl groups, sulfonyl
groups, and combinations of two or more of the foregoing groups. As will be
appreciated by those of ordinary skill in the art, such functional groups may be
constituents of the hydrophilic group itself or may constitute a part of the
phosphorylated polyol which is not identified as the hydrophilic group. The intended
end use of the nanoparticle compositions may impact the choice of such functional
groups.
[0053] As noted, the nanoparticle composition provided by the present invention
typically comprises a transition metal oxide core and a shell comprised of a
phosphorylated polyol. In the product nanoparticle composition the ratio of the shell
to the core may be determined by elemental analysis. From knowledge of the
chemical make up of the metal oxide nanoparticles and their average size before
treatment with the phosphorylated polyol, a calculation can be made of the amount of
phosphorylated polyol per nanoparticulate metal oxide core particle. In one
embodiment, the present invention provides a nanoparticle composition comprising a
nanoparticulate iron oxide core and a phosphorylated polyol shell wherein the molar
ratio of phosphorylated polyol to iron is in a range from about 0.01 to about 0.25. In
an alternate embodiment, the present invention provides a nanoparticle composition
comprising a nanoparticulate tantalum oxide core and a phosphorylated polyol shellwherein the molar ratio of phosphorylated polyol to tantalum is in a range from about
1 to about 2 . In one embodiment, the present invention provides a nanoparticle
composition comprising a nanoparticulate SPIO core, and a phosphorylated polyol
shell wherein the molar ratio of the phosphorylated polyol to the iron in the
nanoparticulate SPIO core is in a range from about 0.01 to 0.25.
[0054] One aspect of the invention relates to methods for making the nanoparticle
compositions. In general, the method for making a nanoparticle composition
comprises contacting a nanoparticulate metal oxide core with a phosphorylated polyol
shell composition of the present invention. The Experimental Section of this
disclosure provides extensive guidance on the preparation of the nanoparticle
composition provided by the present invention. Typically, the contacting is carried
out in a mixture comprising at least one organic solvent and water.
[0055] In one embodiment, the method comprises providing a nanoparticulate metal
oxide core, and disposing a phosphorylated polyol shell on the core. In one or more
embodiments, the step of providing a nanoparticulate metal oxide core comprises
providing a first precursor material comprising a transition metal, the first precursor
material being susceptible to nanoparticulate metal oxide formation. In one
embodiment, the first precursor material may react with an organic acid to generate
the nanoparticulate metal oxide core. The term "reacts" includes mixing two or more
reactants under conditions which allow them to interact. In an alternate embodiment,
the first precursor material may decompose to generate the nanoparticulate metal
oxide core. In another embodiment, the first precursor material may hydrolyze to
generate the nanoparticulate metal oxide core. Thus, in one embodiment
nanoparticulate metal oxide core is provided by hydrolysis of a metal alkoxide in the
presence of an organic acid. For example, nanoparticulate tantalum oxide tantalum
may be prepared by hydrolysis of tantalum ethoxide. The organic acid may be, for
instance, a carboxylic acid such as isobutyric acid. The hydrolysis reaction may be
carried out in the presence of an alcohol solvent, such as 1-propanol or methanol.
Methods for the preparation of nanoparticulate metal oxide particles are well known
in the art and any suitable method for making a nanoparticle core of an appropriate
material may be suitable for use in this method.[0056] The Experimental Section of this disclosure provides detailed guidance on
protocols for disposing a phosphorylated polyol shell on the nanoparticulate metal
oxide core. In one or more embodiments, disposing the shell on the core comprises
providing a second precursor material comprising a phosphorylated polyol or a
precursor thereto. In some embodiments, the precursor to the phosphorylated polyol
may undergo a hydrolysis reaction in the presence of the nanoparticulate metal oxide
core and thereafter attach to the surface of the nanoparticulate metal oxide core. In an
alternate embodiment, the precursor to the phosphorylated polyol can be attached to
the surface of the nanoparticulate metal oxide core and thereafter hydrolyzed.
[0057] As noted, the nanoparticle compositions provided by the present invention
may be used as contrast agents for diagnostic imaging. In such an application, these
nanoparticle compositions are administered to a subject, in some embodiments a
mammalian subject, and then the subject is thereafter subjected to imaging. The
nanoparticle compositions provided by the present invention may be particularly
useful in MR and X-ray imaging though they may also find utility as contrast agents
in ultrasound or radioactive tracer imaging. In addition, the nanoparticle
compositions provided by the present invention may be useful in other areas such as
cell culture infusion.
[0058] In one embodiment, the present invention provides a diagnostic agent
composition suitable for injection into a mammalian subject, and the diagnostic agent
composition comprises a nanoparticle composition and a pharmaceutically acceptable
carrier or excipient. The nanoparticle composition comprises a nanoparticulate metal
oxide and a phosphorylated polyol, the phosphorylated polyol comprising at least two
phosphate groups and one or more hydrophilic groups selected from the group
consisting of polyethylene ether moieties, polypropylene ether moieties, polybutylene
ether moieties, and combinations of two or more of the foregoing hydrophilic
moieties. In one embodiment, the excipient is an optional component of the
diagnostic agent composition. Suitable excipients are illustrated by, but not limited
to, one or more of salts, disintegrators, binders, fillers, and lubricants. In one
embodiment, the pharmaceutically acceptable carrier may be substantially water.[0059] Diagnostic agent compositions provided by the present invention may be
prepared by contacting a nanoparticle composition of the present invention with a
pharmaceutically acceptable carrier and/or excipient.
[0060] In yet another embodiment, the present invention provides a method of
performing diagnostic imaging, the method comprising (a) administering a diagnostic
agent composition of the present invention to a subject in a pharmaceutically
acceptable carrier or excipient; and (b) subjecting the subject to diagnostic imaging,
wherein the diagnostic agent composition acts as a contrast agent. The diagnostic
agent composition may be administered by injection, inhalation, ingestion, parenteral
injection, or intravenous injection.
[0061] When used in diagnostic imaging, particularly of mammalian subjects and
more particularly of human subjects, the diagnostic agent compositions provided by
the present invention, are typically administered as a suspension in a pharmaceutically
acceptable carrier which may (but is not required to) comprise one or more excipients.
If the administration is to be by injection, particularly parenteral injection, the carrier
is typically an aqueous medium that has been rendered isotonic by the addition of
about 150 mM of NaCl, 5% dextrose or combinations thereof. It typically also has an
appropriate (physiological) pH of between about 7.3 and 7.4. The administration may
be intravascular (IM), subcutaneous (SQ) or most commonly intravenous (IV).
However, the administration may also be via implantation of a depot that then slowly
releases the nanoparticles into the subject's blood or tissue. Alternatively, the
administration may be by ingestion for imaging of the GI tract or by inhalation for
imaging of the lungs and airways.
[0062] The administration to human subjects, particularly intravenous administration,
requires that the diagnostic agent composition be non-toxic in the amounts used and
free of any infective agents such as bacteria and viruses and also free of any pyrogens.
Thus, the nanoparticle composition present in the diagnostic agent composition
should be stable to the necessary purification procedures and not suffer degradation in
their hydrophilicity or change in the size of the constituent nanoparticles.[0063] In one embodiment, the present invention provides a diagnostic agent
composition which may be delivered to the site of administration as a stable aqueous
colloidal suspension with the proper osmolality and pH, as a concentrated aqueous
colloidal suspension suitable for dilution prior to administration to a subject. In an
alternate embodiment, the present invention provides a diagnostic agent composition
as a powder, such as obtained by lyophilization, suitable for reconstitution.
[0064] This written description uses examples to disclose the invention, including the
best mode, and also to enable any person skilled in the art to practice the invention,
including making and using any devices or systems and performing any incorporated
methods. The patentable scope of the invention is defined by the claims, and may
include other examples that occur to those skilled in the art. Such other examples are
intended to be within the scope of the claims if they have structural elements that do
not differ from the literal language of the claims, or if they include equivalent
structural elements with insubstantial differences from the literal language of the
claims.
EXPERIMENTAL SECTION
Example 1 : Synthesis of a Nanoparticulate Metal Oxide Core (SPIO)
[0065] To a 20 mL solution of anhydrous benzyl alcohol, 0.706g of iron (III)
acetylacetonate (2 mmol) and 0.414g of 1-phenyl- 1,2-ethanediol (3 mmol) were
added under stirring condition and the resulting mixture was heated at 170°C for 4hrs.
The reaction mixture was cooled to ambient temperature to form a SPIO core solution
containing 5.6 mg of Fe/mL.
Example 2 : Synthesis of 1.2 Bis phosphate PEG 350 1 2BPP350 (5)
[0066] A stirred solution of PEG350 monomethyl ether (35g, 100 mmol) and
triethylamine (20.2 g, 200 mmol) in methylene chloride (200 mL) was cooled to 0°C,
and methane sulfonyl chloride (17. lg, 150 mmol) was added drop-wise. The reaction
was then allowed to warm to room temperature and was stirred for an additional 3h. A
solution of saturated aqueous ammonium chloride (100 mL) was then added and thelayers were separated. The organic layer was washed with saturated aqueous
ammonium chloride (3 x 100 mL), saturated aqueous sodium bicarbonate solution ( 1
x 100 mL), and finally with a saturated aqueous sodium chloride solution ( 1 x 100
mL). The organic solution was then dried over anhydrous sodium sulfate, filtered,
and the solvent removed under reduced pressure to yield 48g of compound 1 as an oil.
(1)
[0067] Freshly powdered potassium hydroxide (2.98g, 53.1 mmol) was added to
anhydrous DMSO (100 mL), and the mixture was stirred for 1 hour under an inert
atmosphere. 1,2-isopropylideneglycerol (2.81g, 21.3 mmol) was then added,
followed by a drop-wise addition of PEG350 mesylate compound 1 (9.1g, 21.3 mmol)
in 50 ml of anhydrous DMSO. The mixture was then heated to 40 °C and stirred for
18 hours under inert atmosphere. The reaction mixture was then cooled to ambient
temperature, diluted with water (200 mL), and extracted with methylene chloride (4 x
200 mL). The combined organic layers were then washed with water (2 x 200 mL)
and concentrated under reduced pressure yielding compound 2 as a yellow oil. 1 H
NMR (400 MHz, CDC13, δ) : 4.3 (1H, m), 4.05-4.2 (2H, m), 3.5-3.75 (32H, m), 3.4
(3H, s), 1.43 (3H, s), 1.37 (3H, s).
(2)
[0068] 1M HC1 in methanol (50 mL) was added to a stirred solution of 2 (8.8g, 21.4
mmol) in methanol (50 mL), and the reaction was stirred for 18h at ambient
temperature. The mixture was then concentrated under reduced pressure and dried
under high vacuum to yield 8g of compound 3 as an oil. 1 H NMR (400 MHz, CDC13,
δ) : 3.95-4.0 (2H, bs), 3.9 (1H, m), 3.55-3.8 (32H, s), 3.4 (3H, s).(3)
[0069] Tetrazole (0.45M in acetonitrile, 32.4 mmol) was added to a solution of
dibenzyl Ν ,Ν -diisopropylphosphoramidite ( 11.19g, 32.4 mmol) in methylene chloride
(300 mL), and the mixture was stirred at ambient temperature for 30 min. Diol
compound 3 (3.0g, 8.1 mmol) was then added and the mixture was stirred for 18h at
ambient temperature. The reaction was then cooled to -78 °C and m-
chloroperoxybenzoic acid (77%) (59g, 32.4 mmol) was added as a single portion. The
reaction mixture was then stirred at -78°C for 10 minutes, allowed to warm to room
temperature and then stirred for an additional 4h. A 10% (w/v) aqueous solution of
sodium sulfite (100 mL) was then added and the layers were separated. The aqueous
layer was back extracted with methylene chloride (100 mL) and the combined organic
extracts were evaporated under reduced pressure. The resulting yellow oil was
purified using column chromatography (hexanes : ethyl acetate) followed by a solvent
change (methylene chloride : methanol) yielding 4.58g of compound 4 . 1 H MR
(400 MHz, CDC13, δ) : 7.28-7.35 (20H, m), 5.0-5.1 (8H, m), 4.7 (1H, m), 4.1-4.25
(2H, m), 3.55-3.8 (32H, m), 3.4 (3H, s).
(4)
[0070] Palladium on carbon (10%, 3g) was added to a solution of compound 4 (4.58g,
5.14 mmol) in ethanol (100 mL) and the mixture was stirred at ambient temperatureunder an H2 atmosphere for 2 days. The reaction mixture was then filtered through
celite and the filter cake was washed with ethanol (2 x 50 mL). The filtrate was
evaporated under reduced pressure yielding 6g of compound 5 as a waxy solid. 1 H
MR (400 MHz, D20, δ) : 4.38 (1H, bs), 3.9-4.0 (2H, m), 3.5-3.7 (32H, m), 3.27 (3H,
s).
(5)
Example 3 : Synthesis of Nanoparticle Composition (VII) (1.2BPP350 SPIO)
[0071] PEG350 Bisphosphate compound 5 (1.06 g, 2 mmol) was dissolved in 200mM
aqueous sodium hydroxide solution (20 mL). THF (20 mL) was then added, and the
pH of the solution was adjusted to 8 by drop-wise addition of 3M sodium hydroxide.
A solution of SPIO cores in benzyl alcohol (10 mL of the 5.6mg Fe/mL solution) was
then added, and the solution was stirred overnight at 50 °C. The reaction was then
cooled to ambient temperature and diluted with hexanes (50 mL). The layers were
separated and the aqueous layer was purified by tangential flow filtration (50K
MWCO membrane washed against 4 L of water) to provide a stable suspension of the
nanoparticle composition VII. The final particles had a hydrodynamic diameter of 9
nM as measured in a 150 mM sodium chloride solution by dynamic light scattering.
The size of the particles did not change after 2 days in the 150 mM sodium chloride
solution incubated at 40 °C.
Example 4 : Synthesis of L2BPP350 Tantalum Oxide
[0072] Water (0.1 1 mL) was added to a stirred solution of compound 5 (3.92 g, 7.4
mmol) dissolved in anhydrous methanol (75 mL), and the solution was stirred for 20
minutes. Tantalum ethoxide (1.5 g, 3.69 mmol) was then added drop-wise, the
mixture was stirred at ambient temperature for 1 h, and then heated at 50 °C for 18h.The reaction was then cooled to ambient temperature and diluted with water (250
mL). The pH was adjusted to ~8 by the addition of ammonium hydroxide, the solution
was concentrated until the methanol was fully evaporated, and the remaining aqueous
solution was passed through a 100 nm filter. The particles were purified using dialysis
(3.5K MWCO PES membrane washed against 1 L of water with 4 exchanges). The
retained solution was then passed through a 100 nm filter yielding particles having a
hydrodynamic size of 4.7 nM as measured in water by dynamic light scattering.
Example 5 : Synthesis of 1.2BPP440 (10)
[0073] A solution of monodisperse decaethylene glycol monomethyl ether
(Biomatrik; Zhejiang, China) (lOg, 2 1 mmol) and triethylamine (3.85 g, 38 mmol) in
methylene chloride (200 mL) was cooled to -30°C, and methane sulfonyl chloride
(3.64 g, 31.7 mmol) was added drop-wise. The reaction was allowed to warm to 0 °C
over 3h. Saturated aqueous ammonium chloride (100 mL) was then added and the
layers were separated. The aqueous layer was back extracted with methylene chloride
(50 mL), the combined organics washed with a saturated aqueous sodium bicarbonate
solution ( 1 x 100 mL), dried over magnesium sulfate, filtered, and the solvent
removed under reduced pressure to yield 12g of compound 6 as an oil.
(6)
[0074] Freshly powdered potassium hydroxide (3.04 g, 54.3 mmol) was added to
anhydrous DMSO (200 mL), and the mixture was stirred for 1.5 hours under an inert
atmosphere. A solution of 1,2-isopropylideneglycerol (2.87 g, 21.7 mmol) and
PEG440 mesylate compound 6 (12.0 g, 21.7 mmol) in 20 ml of anhydrous DMSO
was added, and the mixture was stirred for 18 hours at 40 °C under inert atmosphere.
The reaction mixture was then cooled to ambient temperature, diluted with water (250
mL) and extracted with methylene chloride (2 x 500 mL). The combined organic
layers were then washed with water ( 1 x 500 mL) and concentrated under reduced
pressure yielding compound 7 as a light yellow oil. 1 H MR (400 MHz, CDC13, δ) :4.3 (1H, m), 4.05-4.1 (1H, m), 3.7-3.8 (2H, m), 3.6-3.7 (39H, m), 3.5-3.6 (4H, m), 3.4
(3H, s), 1.4 (6H, d).
(7)
[0075] IN HC1 in methanol (50 mL) was added to a stirred solution of 7 (9.17 g, 15.6
mmol) in methanol (50 mL). The reaction was stirred for 18h at ambient temperature,
then concentrated under reduced pressure and dried under high vacuum to yield 8.8 g
of compound 8 as an oil. 1 H NMR (400 MHz, CDC13, δ) : 3.9 (1H, m), 3.65-3.8
(40H, s), 3.55-3.65 (4H, m), 3.4 (3H, s).
(8)
[0076] Tetrazole (0.45M in acetonitrile, 22 mmol) was added to a solution of dibenzyl
N,N-diisopropylphosphoramidite (7.62 g, 22 mmol) in methylene chloride (300 mL),
and the mixture was stirred at ambient temperature for 30 min. Diol compound 8 (3.0
g, 5.5 mmol) was then added, the mixture was stirred for 18h at ambient temperature.
The reaction was then cooled to -78 °C and m-chloroperoxybenzoic acid (77%) (3.81
g, 22 mmol) was added as a single portion. The reaction mixture was then stirred at -
78 °C for 10 minutes, allowed to warm to room temperature and then stirred for an
additional 4h. A 10% (w/v) aqueous solution of sodium sulfite (100 mL) was then
added and the layers were separated. The aqueous layer was back extracted with
methylene chloride (100 mL) and the combined organic extracts were evaporated
under reduced pressure. The resulting oil was purified using column chromatography
(hexanes : ethyl acetate) followed by a solvent change (methylene chloride :
methanol) yielding 1.56 g of compound 9 . 1 H NMR (400 MHz, CDC13, δ) : 7.3-7.4(20H, m), 5.0-5.1 (8H, m), 4.7 (1H, m), 4.1-4.25 (2H, m), 3.55-3.7 (42H, m), 3.4 (3H,
s).
[0077] Palladium on carbon (10%) (0.25 g) was added to a stirred solution of
compound 9 (1.56 g, 1.46 mmol) in ethanol (100 mL), and the mixture was stirred at
ambient temperature under an H2 atmosphere for 2 days. The reaction mixture was
then filtered through celite and the filter cake washed with ethanol (2 x 50 mL). The
filtrate was evaporated under reduced pressure yielding 1.03 g of compound 10 as a
clear oil. 1 H MR (400 MHz, D20 , δ) : 4.395 (1H, m), 3.9-4.0 (2H, m), 3.5-3.65
(42H, m), 3.25 (3H, s).
(10)
Example 6 : Synthesis of Nanoparticle Composition (VIII) (1.2BPP440 SPIO)[0078] 1M aqueous sodium hydroxide (3 mL) was added to a stirred solution of
compound 10 (0.71 g, 2 mmol) dissolved in THF (20 mL) and water (15 mL). A
solution of SPIO cores in benzyl alcohol (10 mL of the 5.6mg Fe/mL solution) was
then added, and the mixture was stirred overnight at 50 °C. The reaction was then
cooled to ambient temperature and diluted with hexanes (2 x 50 mL). The layers were
separated and the aqueous layer was then purified by tangential flow filtration (3OK
MWCO membrane washed against 4 L of water) to provide a stable suspension of the
nanoparticle composition VIII. The final particles had a hydrodynamic diameter of
10.3 nM as measured in water by dynamic light scattering. The size of the particles
did not change after 2 days in 150 mM sodium chloride solution incubated at 40 °C.
Example 7 : Synthesis of a 1.2BPP750 (15)
[0079] A solution of PEG750 monomethyl ether (75g, 100 mmol) and triethylamine
(30. 3g, 300 mmol) in methylene chloride (700 mL) was cooled to 0°C, and methane
sulfonyl chloride (22. 8g, 200 mmol) was added drop-wise. The resulting reaction was
allowed to warm to room temperature and then stirred for an additional 3h. A solution
of saturated aqueous ammonium chloride (200 mL) was then added and the layers
were separated. The organic layer was washed with saturated aqueous ammonium
chloride (4 x 200 mL), saturated aqueous sodium bicarbonate solution ( 1 x 200 mL),
and finally with a saturated aqueous sodium chloride solution ( 1 x 200 mL). The
organic solution was then dried over anhydrous sodium sulfate, filtered, and the
solvent removed under reduced pressure to yield 84g of compound 11 as an oil. 1 H
MR (400 MHz, CDC13, δ) : 4.36 (2H, m), 3.75 (2H, m), 3.62 (64H, br. s), 3.55 (2H,
m), 3.35 (3H, 3), 3.07 (3H, s).
( 11)
[0080] Freshly powdered potassium hydroxide (12.75g, 225 mmol) was added to
anhydrous DMSO (150 mL), and the mixture was stirred for 30 minutes under an
inert atmosphere. 1,2-isopropylideneglycerol (26.4g, 200 mmol) was then added,followed by a drop-wise addition of PEG mesylate compound 11 (84g, 100 mmol) in
500 ml of anhydrous DMSO. The mixture was stirred for three days under inert
atmosphere. A mixture of 80% aqueous sodium chloride (700 mL) and methylene
chloride (500 mL) was then added, the layers were separated, and the aqueous layer
was back-extracted with methylene chloride (4 x 300 mL). The combined organic
layers were then washed with saturated sodium chloride ( 1 x 500 mL), dried over
anhydrous sodium sulfate, and filtered. The solvent was removed under reduced
pressure, and the remaining DMSO was distilled off under high vacuum. The
material was then dissolved in warm THF (200 mL) and heptane (75 mL), a small
amount of solid was filtered off, and the filtrate was allowed to crystallize overnight
at 5°C. At this time, cold heptane (200mL) was added, the solid was collected via
cold filtration. Residual 1,2-isopropylideneglycerol was removed by dissolving the
material in water (700 mL) and washing with heptane (5 x 150 mL), yielding product
compound 12 in aqueous solution. The solvent was removed from a small aliquot of
the aqueous solution, yielding solid compound 12. 1 H MR (400 MHz, CDC13, δ) :
4.15 (1H, m), 3.92 (1H, m), 3.3-3.7 (68H, m), 3.25 (3H, s), 1.25 (6H, d).
(12)
[0081] The resulting aqueous solution of compound 12 (700 mL) was mixed with 3N
HC1 (100 mL), and stirred overnight. The majority of the water was then stripped off
via rotary evaporation, and the remaining material was suspended in methylene
chloride (600 mL). Solid anhydrous sodium carbonate (50g) was then added
cautiously, and the mixture stirred for 1 hour. The solid was filtered off, and the
solvent removed via rotary evaporation. Toluene (300 mL) was then added and the
solution was refluxed for 2 hours with a Dean Stark trap to remove any remaining
water. The solution was then cooled to room temperature, the toluene was stripped
off by rotary evaporation, and the remaining material was dried under hi vacuum toyield 72g of compound 13 as an oily solid. 1 H NMR (400 MHz, CDC13, δ) : 3.78 (2H,
m), 3.45-3.7 (68H, m), 3.35 (3H, s).
(13)
[0082] Tetrazole (0.45M in acetonitrile, 72.8 mmol) was added to a solution of
dibenzyl N,N-diisopropylphosphoramidite (25. lg, 72.8 mmol) in methylene chloride
(200 mL), and the mixture was stirred at ambient temperature for 30 min. Diol
compound 13 was then added (15g, 18.2 mmol) and the resulting mixture was stirred
for 18h at 40 °C. The reaction was then cooled to -35°C, and m-chloroperoxybenzoic
acid (77%) (12.6g, 72.8 mmol) was added as a single portion. The reaction was then
stirred at -35 °C for 5 min, allowed to warm to room temperature, and then stirred for
an additional 4h. A 10% (w/v) aqueous solution of sodium sulfite (100 mL) was then
added, the reaction was stirred for 30 min, the layers were separated, and the organic
layer evaporated under reduced pressure. The resulting yellow oil was purified using
column chromatography (hexanes : ethyl acetate) followed by a solvent change
(methylene chloride : methanol) yielding 8g of compound 14 as a clear oil. 1 H NMR
(400 MHz, CDC13, δ) : 7.3-7.4 (20H, m), 5.0-5.1 (8H, m), 4.65 (1H, m), 4.1-4.25 (2H,
m), 3.55-3.8 (70H, m), 3.4 (3H, s).
(14)[0083] Palladium on carbon (10%) (0.5g) was added to a stirred solution of
compound 14 (8g, 5.8 mmol) in ethanol (100 mL), and the mixture was stirred at
ambient temperature under an H2 atmosphere for 2 days. The reaction mixture was
then filtered through celite and the filter cake was washed with ethanol (2 x 50 mL).
The filtrate was evaporated under reduced pressure yielding 6g of compound 15 as a
waxy solid. 1 H NMR (400 MHz, CDC13, δ) : 4.75 (1H, bs), 4.1-4.3 (2H, m), 3.55-3.8
(70H, m), 3.4 (3H, s).
(15)
Example 8 : Synthesis of Nanoparticle Composition (IX) (1.2BPP750 SPIO)
[0084] 1M aqueous sodium hydroxide solution (0.6 mL) was added to a stirred
solution of PEG750 bisphosphate compound 15 (0.203 g, 0.2 mmol) dissolved in THF
(4 mL) and water (2.5 mL). A solution of SPIO cores in benzyl alcohol (4 mL of a
2.8mg Fe/mL solution) was then added, and the solution was stirred overnight at 50
°C. The reaction was then cooled to ambient temperature and diluted with hexanes
(10 mL). The layers were separated and the aqueous layer was then purified using
centrifuge filters (3OK MWCO washed against water) to provide a stable suspension
of the nanoparticle composition IX. The final particles had a hydrodynamic diameter
of 13 nM as measured in a 150 mM sodium chloride solution by dynamic light
scattering. The size of the particles did not change after 2 days in 150 mM sodium
chloride solution incubated at 40 °C. The material could be sterilized by autoclave
(121°C, 15 minutes, 5% mannitol formulation) with no sign of aggregation or change
in particle size.
Example 9 : Synthesis of a 1.2BPP2000 (20)[0085] A solution of PEG1900 monomethyl ether (95g, 50 mmol) and triethylamine
(20.2g, 200 mmol) in methylene chloride (700 mL) was cooled to 0°C, and methane
sulfonyl chloride (17. lg, 150 mmol) was added drop-wise. The resulting reaction was
allowed to warm to room temperature and then stirred for an additional 18h. A
solution of saturated aqueous ammonium chloride (200 mL) was then added and the
layers were separated. The organic layer was washed with saturated aqueous
ammonium chloride (4 x 200 mL), saturated aqueous sodium bicarbonate solution ( 1
x 200 mL), and finally with a saturated aqueous sodium chloride solution ( 1 x 200
mL). The organic solution was then dried over anhydrous sodium sulfate, filtered,
and the solvent removed under reduced pressure, yielding lOOg of compound 16 as a
white solid. 1 H MR (400 MHz, CDC13, δ) : 4.38 (2H, m), 3.75 (2H, m), 3.62 (176H,
br. s), 3.55 (2H, m), 3.38 (3H, 3), 3.10 (3H, s). .
(16)
[0086] Freshly powdered potassium hydroxide ( 11.2g, 200 mmol) was added to
anhydrous DMSO (150 mL), and the mixture was stirred for 30 minutes under an
inert atmosphere. 1,2-isopropylideneglycerol (26.4g, 200 mmol) was then added,
followed by a drop-wise addition of PEG2000 mesylate compound 16 (lOOg, 50
mmol) in 500 ml of anhydrous DMSO. The mixture was then stirred for three days
under inert atmosphere. 80% aqueous sodium chloride (700 mL) and methylene
chloride (500 mL) were then added, the layers were separated, and the aqueous layer
was back-extracted with methylene chloride (4 x 300 mL). The combined organic
solution was then washed with saturated sodium chloride ( 1 x 500 mL), dried over
anhydrous sodium sulfate, and filtered. The solvent was removed under reduced
pressure, and the remaining DMSO was distilled off under high vacuum. The
material was then dissolved in hot THF (300 mL) and heptane (100 mL), a small
amount of solid was filtered off, and the filtrate was allowed to crystallize overnight
at 5°C. Cold heptane (200 mL) was then added, and the solid was collected via cold
filtration. The solid was recrystallized a second time from comparable amounts ofsolvent, and the product was dried yielding 86g of compound 17 as a solid. 1 H MR
(400 MHz, CDC13, δ) : 4.25 (1H, m), 4.00 (1H, m), 3.4-3.8 (172H, m), 3.33 (3H, s),
1.35 (6H, d)
(17)
[0087] 3N HC1 (100 mL) was added to a stirred solution of compound 17 (86 gm) in
water (600 mL), and the mixture was stirred overnight. The majority of the water was
then stripped off via rotary evaporation, and the remaining material was suspended in
methylene chloride (600 mL). Solid anhydrous sodium carbonate (50g) was then
cautiously added, and the mixture stirred for 1 hour. The solid was then filtered off,
and the solvent was removed from the filtrate via rotary evaporation. Toluene (300
mL) was then added and the mixture was refluxed for 2 hours with a Dean Stark trap
to collect any remaining water. The mixture was then cooled to room temperature,
the toluene was removed under reduced pressure, and the remaining material was
dried under hi vacuum, yielding 75g of compound 18 as a solid. 1 H MR (400 MHz,
CDCI
3, δ) : 3.78 (2H, m), 3.45-3.7 (185H, m), 3.30 (3H, s).
(18)
[0088] Tetrazole (0.45M in acetonitrile, 40.5 mmol) was added to a solution of
dibenzyl Ν ,Ν -diisopropylphosphoramidite (14g, 40.5 mmol) in methylene chloride
(200 mL), and the mixture was stirred at ambient temperature for 30 min. Diol
compound 18 was added (20g, 10.1 mmol) and the resulting mixture was stirred for
2d at 40 °C. The reaction was then cooled to -35°C, and m-chloroperoxybenzoic acid
(77%) (6.98g, 40.5 mmol) was added as a single portion. The reaction was stirred at -
35 °C for 5 min, then allowed to warm to room temperature and stir for an additional4h. A 10% (w/v) aqueous solution of sodium sulfite (100 mL) was then added, the
reaction was stirred for 30 min, the layers separated, and the organic layer was
evaporated under reduced pressure. The resulting yellow oil was purified using
column chromatography (hexanes : ethyl acetate) followed by a solvent change
(methylene chloride : methanol) yielding 15g of compound 19 as a solid. 1 H NMR
(400 MHz, CDC13, δ) : 7.29-7.35 (20H, m), 5.0-5.1 (8H, m), 4.65 (1H, m), 4.1-4.25
(2H, m), 3.5-3.75 (184H, m), 3.4 (3H, s).
(19)
[0089] Palladium on carbon (10%) (0.5g) was added to a stirred solution of
compound 19 (15g, 5.9 mmol) in ethanol (100 mL), and the mixture was stirred at
ambient temperature under an H2 atmosphere for 2d. The reaction mixture was then
filtered through celite and the filter cake washed with ethanol (2 x 50 mL). The filtrate
was evaporated under reduced pressure yielding 11.8g of compound 20 as a waxy
solid. 1 H NMR (400 MHz, CDC13, δ) : 4.75 (1H, bs), 4.2-4.3 (2H, m), 3.55-3.85
(184H, m), 3.4 (3H, s).
(20)Example 10: Synthesis of Nanoparticle Composition (X) (1.2BPP2000 SPIO)
[0090] A 1M aqueous sodium hydroxide solution (0.6 mL) was added to a stirred
solution of PEG2000 bisphosphate compound 20 (0.440 g, 0.2 mmol) in THF (4 mL)
and water (2.5 mL). A solution of SPIO cores in benzyl alcohol (4 mL of a 2.8mg
Fe/mL solution) was then added, and the mixture was stirred overnight at 50 °C. The
reaction was then cooled to ambient temperature and diluted with hexanes (10 mL).
The layers were separated and the aqueous layer was then purified via centrifuge
filtration (3OK MWCO washed against water) to provide a stable suspension of the
nanoparticle composition X. The final particles had a hydrodynamic diameter of 16
nM as measured in a 150 mM sodium chloride solution by dynamic light scattering.
The size of the particles did not change after 2 days in 150 mM sodium chloride
solution incubated at 40 °C. The material could be sterilized by autoclave (121°C, 15
minutes) with no sign of aggregation or change in particle size
Example 11: Synthesis of a Mannitol-Based Phosphorylated polyol "P2P4Man" (23)
[0091] Freshly powdered potassium hydroxide (0.47g, 8.4 mmol) in anhydrous
DMSO (30mL) was stirred for 30 minutes under an inert atmosphere. Mannitol
(0.1 82g, 1 mmol) was then added, followed by PEG440 mesylate compound 6 (2.2g,
4 mmol). The mixture was stirred for three days under inert atmosphere. 80%
saturated aqueous sodium chloride (100 mL) and methylene chloride (100 mL) were
then added, the layers were separated, and the aqueous layer was back-extracted with
methylene chloride (4 x 75 mL). The combined organic solution was then washed
with saturated sodium chloride ( 1 x 100 mL), dried over anhydrous sodium sulfate,
and filtered. The filtrate was removed under reduced pressure, and the remaining
DMSO was distilled off under high vacuum yielding 2.3g of compound 2 1 as a thick
oil. 1 H MR (400 MHz, CDC13, δ) : 3.5-3.9 (44H, m), 3.39 (3H, s) indicated structure
2 1 wherein the groups O-R2 are principally hydroxy groups and PEG groups
0 (CH2CH20 )ioCH3 and wherein the ratio of hydroxy groups to PEG groups is
approximately 2.2 to 3.8.(21)
[0092] Tetrazole (0.45M in acetonitrile, 4 mmol) was added to a solution of dibenzyl
Ν,Ν-diisopropylphosphoramidite (1.38g, 4 mmol) in methylene chloride (15 mL), and
the mixture was stirred at ambient temperature for 30 min. Diol compound 2 1 (2.3g, 1
mmol) in 25 mL of methylene chloride was then added, and the resulting mixture was
stirred for 3d at ambient temperature under an inert atmosphere. The solution was
then cooled to -78 °C and m-chloroperoxybenzoic acid (77%) (0.9g, 4 mmol) in 10
mL of methylene chloride was added. The reaction was then allowed to warm to
room temperature over 2h with stirring. A 10% (w/v) aqueous solution of sodium
sulfite (20 mL) was then added, the reaction was stirred for 30 min, the layers were
separated and the aqueous layer was back extracted with methylene chloride (2 x 20
mL). The combined organic layers were washed with saturated sodium chloride (50
mL), dried over sodium sulfate, filtered and the filtrate evaporated under reduced
pressure. The resulting product was purified using column chromatography (hexanes :
ethyl acetate) followed by a solvent change (methylene chloride : methanol) yielding
0.7g of compound 22 the structure of which was confirmed by 1 H NMR (400 MHz,
CDC13, δ) : 7.25 (5.85H, m), 5.0 (2.34H, m), 3.9-3.5 (43H, m), 3.39 (3H, 3). NMR
integration indicated that the groups O-R2 were principally dibenzyl phosphate groups
(PhCH20 )
2P0 2 groups and PEG groups O(CH2CH2O)i
0CH3, and that the ratio of
phosphorus to PEG was 0.58, with approximately 3.8 PEG groups and approximately
2.2 dibenzyl phosphate groups per molecule.(22)
[0093] A solution of compound 22 (0.7g) in methanol (20 mL) was sparged with
nitrogen for 2 minutes, then palladium on carbon (10%) (0.07g) was added. The
reaction was stirred at ambient temperature under an H2 atmosphere for 18h, after
which time TLC analysis indicated that the reaction was complete. The reaction
mixture was filtered through celite and the filtrate was evaporated under reduced
pressure yielding 0.59g of compound 23. 1 H MR (400 MHz, CDC13, δ) : 4.2-3.5
(44H, m), 3.40 (3H, 3) indicated that the groups O-R2 were principally phosphate
groups (ΗΟ)
2Ρ0 2 groups and PEG groups 0(CH 2CH20)ioCH 3, with approximately
3.8 PEG groups and approximately 2.2 phosphate groups per molecule.
(23)
Example 12: Synthesis of Nanoparticle composition "P2P4Man" SPIO
[0094] A reaction vial was charged with water (2 mL), compound 23 (0.214g) and 5N
NaOH (50 uL). The contents were shaken until fully dissolved, yielding a solution
with pH = 8.0. The mixture was then lyophilized, and the residue was dissolved in
THF (10 mL). A solution of SPIO core in benzyl alcohol (2.3 mL, 5.5 mg Fe/mL)
was then added, and the solution was capped and stirred overnight at 50 °C. Water (6
mL) was then added, the mixture was shaken, and the dark color transferred
completely into the aqueous layer. The layers were separated, and the aqueous layer
solution was washed with hexane (2 mL) and filtered through a 20 nm filter. The
solution was then syringed into a 3500 MW dialysis cassette, and the dialyzed against
water ( 1 L) for 24 hours, changing the dialysis bath water 4 times over the course of
the dialysis to provide a stable suspension of nanoparticle composition wherein R2 is .
The final particles had a hydrodynamic diameter of 11 nM as measured in a 150 mMsodium chloride solution by dynamic light scattering. The size of the particles did not
change after 3 days in the 150 mM sodium chloride solution incubated at 40 °C.
Example 13: Synthesis of a 1.3BPP350 (27)
[0095] Freshly powdered potassium hydroxide (1.03 g, 18.4 mmol) in anhydrous
DMSO was stirred for 1 hour under an inert atmosphere. l,3-dibenzyloxy-2-propanol
(2.0 g, 7.34 mmol) and PEG350 mesylate compound 1 (3.14 g, 7.34 mmol) in 15 ml
of anhydrous DMSO were then added, and the mixture was stirred at 40 °C for 18
hours under an inert atmosphere. The reaction mixture was then cooled to ambient
temperature, diluted with water (100 mL) and extracted with methylene chloride (2 x
150 mL). The combined organic layers were then washed with water (2 x 50 mL) and
concentrated under reduced pressure yielding compound 24 as a yellow oil.
(24)
[0096] Palladium on carbon (10%) (0.41 g) was added to a stirred solution of
compound 24 (4.5 g, 7.34 mmol) in dry methanol (150 mL), followed by an 88%
formic acid solution (5 mL). The mixture was then stirred for 18h at ambient
temperature. The reaction mixture was then filtered through celite and the filter cake
washed with methanol (2 x 50 mL). The filtrate was evaporated under reduced
pressure yielding 2.7 g of compound 25 as a clear oil. 1 H MR (400 MHz, CDC13, δ) :
3.8-3.9 (2H, m), 3.5-3.8 (32H, m), 3.4 (3H, s).
(25)[0097] Tetrazole (0.45M in acetonitrile, 29.4 mmol) was added to a solution of
dibenzyl N,N-diisopropylphosphoramidite (10.14 g, 29.4 mmol) in methylene
chloride (200 mL), and the mixture was stirred at ambient temperature for 30 min.
Diol compound 25 (2.7 g, 7.34 mmol) was then added, and the resulting mixture was
stirred for 18h at ambient temperature. The reaction mixture was then cooled to -78
°C, and m-chloroperoxybenzoic acid (77%) (5.07 g, 29.4 mmol) was then added as a
single portion. The mixture was stirred at -78 °C for 10 minutes, allowed to warm to
room temperature and stirred for an additional 4h. A 10% (w/v) aqueous solution of
sodium sulfite (100 mL) was then added and the layers were separated. The aqueous
layer was back extracted with methylene chloride (100 mL) and the combined organic
extracts were evaporated under reduced pressure. The resulting product was utilized
without further purification as yellow oil compound 26. 1 H NMR (400 MHz, CDC13,
δ) : 7.3-7.45 (20H, m), 4.98-5.1 (8H, m), 3.95-4.2 (4H, m), 3.5-3.7 (28H, m), 3.4 (3H,
s).
(26)
[0098] Palladium hydroxide (0.2 g) was added to a stirred solution of compound 26
(4.18g, 4.69 mmol) in ethanol (100 mL), and the mixture was stirred at ambient
temperature under an H2 atmosphere for 2 days. The reaction mixture was then
filtered through celite and the filter cake was washed with ethanol (2 x 50 mL). The
filtrate was then evaporated under reduced pressure yielding 2.5 g of compound 27 as
a clear oil. 1 H NMR (400 MHz, CDC13, δ) : 3.85-4.0 (3H, m), 3.7-3.8 (2H, m), 3.5-
3.65 (28H, m), 3.27 (3H, s).(27)
Example 14: Synthesis of Nanoparticle Composition (XVI) (1.3BPP350 SPIO)
[0099] THF ( 1 mL) was added to a stirred solution of compound 27 (0.106 g, 0.2
mmol) in 200mM aqueous sodium hydroxide solution ( 1 mL). The pH was then
adjusted to 8 by drop-wise addition of a 3M sodium hydroxide solution. A solution of
SPIO cores in benzyl alcohol ( 1 mL of a 5.6mg Fe/mL solution) was then added, and
the mixture was stirred overnight at 50 °C. The reaction was then cooled to ambient
temperature, the layers separated and the aqueous layer was then purified using
dialysis (10K MWCO PES membrane washed against 1 L of water with 4 exchanges).
The retained solution was then passed through a 100K MWCO centrifuge membrane
to remove any remaining aggregates to provide a stable suspension of nanoparticle
composition XVI. The final particles had a hydrodynamic diameter of 9.5 nM as
measured in a 150 mM sodium chloride solution by dynamic light scattering.
Example 15: Synthesis of a 1.3BPP2000 (31)
[00100] Freshly powdered potassium hydroxide (960 mg, 17.1 mmol) in
anhydrous DMSO (10 mL) was stirred for 20 min. under an inert atmosphere. 2-
Phenyl-l,3-dioxan-5-ol (3.08 gm g, 17.1 mmol) and PEG2000 mesylate compound 16
(8.55 g, 4.27 mmol) in DMSO (80 mL) were then added, and the mixture was stirred
for 18 hours at room temperature under an inert atmosphere. A 90% saturated sodium
chloride solution (150 mL) and methylene chloride (100 mL) were then added, and
the layers were separated. The aqueous layer was extracted with methylene chloride
(2 x 75 mL). The combined organic layers were then washed with saturated sodium
chloride (75 mL), dried over anhydrous sodium sulfate, filtered, and then concentrated
under reduced pressure yielding compound 28 as a yellow oily solid. Excess DMSOwas distilled off under hi vacuum, and the remaining solid was recrystallized from a
mixture of hot THF (100 mL) and hot hexane (40 mL). 1 H NMR (400 MHz, CDC13,
δ) : 7.50 (2H, m), 7.38 (3H, m) 5.58 (1H, s), 4.38 (2H, m), 4.05 (2H, m), 3.9-3.5
(176H, m), 3.39 (3H, s).
(28)
[0101] Palladium hydroxide (0.5 g) was added to a stirred solution of compound 28
(4.18g, 4.69 mmol) in ethanol (100 mL), and the mixture was stirred at ambient
temperature under an H2 atmosphere for 18 hours. The mixture was then filtered
through celite and the filter cake was washed with ethanol (2 x 50 mL). The filtrate
was evaporated under reduced pressure yielding 5.0 g of compound 29 as a white
sticky solid. 1 H NMR (400 MHz, CDC13, δ) : 3.85 (3H, m), 3.5-3.75 (178H, m), 3.38
(3H, s).
(29)
[0102] Tetrazole (0.45M in acetonitrile, 10.1 mmol) was added to a solution of
dibenzyl N,N-diisopropylphosphoramidite (3.5 g, 10.1 mmol) in methylene chloride
(100 mL), and the mixture was stirred at ambient temperature for 30 min. Diol
compound 29 was then added (5.0 g, 2.53 mmol) and the resulting mixture was stirred
for 48h at 40 °C. The reaction was then cooled to -35 °C and tert-butylhydroperoxide
(90%) (0.91 g, 10.1 mmol) was added as a single portion. The reaction mixture was
then stirred at -35 °C for 10 minutes, allowed to warm to room temperature, and thenstirred for an additional 4h. A 10% (w/v) aqueous solution of sodium sulfite (100 mL)
was then added and the layers were separated. The aqueous layer was back extracted
with methylene chloride (100 mL) and the combined organic extracts were evaporated
under reduced pressure. The resulting product was utilized without further
purification as yellow oil compound 30.
(30)
bisdibenzyl phosphate 30 was converted to bisphosphate 3 1 as taught in Example 13
herein.
(31)
Comparative Example 1 : Synthesis of a PEG2000 monophosphate (33)
[0103] A solution of PEG1900 monomethyl ether in methylene chloride containing
an excess of triethylamine and catalytic 4-dimethylaminopyridine (DMAP) was
treated with an excess of diphenyl chlorophosphate. The mixture was stirred for 24
hours under nitrogen, quenched by the addition of an excess of IN hydrochloric acid,and the layers were separated. The organic layer was washed once with water, the
solvent was distilled off first at atmospheric pressure, then at reduced pressure, to
azeoptropically remove any remaining water. The residue was crystallized from a
mixture of hot THF and hexanes, then was washed with methyl t r t-butyl ether. After
drying under vacuum, the product was redissolved in tetrahydrofuran and treated with
activated charcoal. The charcoal was filtered off, and the solution was diluted with
hexanes, cooled, and the precipitated product collected by filtration. The solids were
washed with methyl tert-butyl ether and hexanes, then dried under vacuum for a 70-
90% yield of product PEG2000 monophosphate diphenyl ester 32.
(32)
[0104] A solution of the PEG2000 monophosphate diphenyl ester 32 in acetic acid
was hydrogenated at 45 °C and 2-4 bar pressure in the presence of 5 mole% platinum
oxide until the hydrogen uptake ceased. After cooling, the catalyst was removed by
filtration, the filtrate was concentrated under reduced pressure and the residue was
dissolved in tetrahydrofuran. Hexanes were added, the mixture was cooled, the
precipitated product was collected by filtration, and the solid was washed with methyl
ter t-butyl ether and hexanes. The product monophosphate 33 was then dried under
vacuum at ambient temperatures for a 70-90% yield of product. This material was not
stable to autoclave sterilization, whereas the 1,2 and 1,3-BPP2000 materials were
shown to be stable under comparable autoclave conditions.
(33)
Comparative Example-2: Synthesis of a PEG2000 monophosphate coated SPIO[0105] PEG2000 monophosphate (14.57 g, 7.0 mmol) prepared in Comparative
Example 1was suspended in THF (161 mL) and a solution of SPIO nanoparticles (35
mL at 5.6 mg Fe/mL in benzyl alcohol) was added. The resulting suspension was
stirred at 50 °C for 16 h during which the reaction became homogeneous. The
reaction was then cooled to room temperature and diluted with water (200 mL). The
resulting layers were separated and the aqueous layer was washed with hexanes (2 x
200 mL). The remaining volatiles were removed from the aqueous layer in vacuo and
the resulting aqueous nanoparticle suspension was washed against a 100 kDa MWCO
tangential flow filtration membrane with water (3.75 L) to provide a suspension of
The resulting nanoparticles had a hydrodynamic diameter of 18.7 nm in 150 mM
NaCl at 25 °C as measured by dynamic light scattering.
Comparative Example 3 : Autoclave stability of PEG2000monophosphate coated
SPIO
[0106] A suspension of PEG2000monophosphate coated SPIO nanoparticles prepared
in Comparative Example 2 ( 1 mL at 10 mg Fe/mL) in water were autoclaved in a
closed, 2 dram glass vial at 121 °C and 20 atm for 15 min. After the autoclave cycle
was complete, all color had precipitated from the solution, indicating complete
aggregation of the nanoparticles.
Comparative Example 4 : Synthesis of a PEG350 monophosphate coated SPIO (XVII)
[0107] A solution of PEG-350 mono(methyl ether) (8.54 g, 24.4 mmol) dissolved in
CH2CI2 (80 mL) was charged with triethyl amine (3.68 g, 36.6 mmol) followed by 4-
N,N-dimethylaminopyridine (0.298 g, 2.44 mmol). The resulting solution was cooled
to 0 °C, diphenyl chlorophosphate (7.87 g, 29.3 mmol) was added dropwise, and the
reaction was stirred at 0 °C for 10 min. The reaction was then warmed to room
temperature and stirred for an additional 16 h . The reaction was quenched by the
addition of 10% HC1 (80 mL) and the resulting layers were separated. The organic
layer was washed with water (80 mL) and brine (80 mL) and dried over anhydrous
MgS0 4. Filtration and removal of the solvent in vacuo afforded the mono phosphate
diphenyl ester of PEG-350 mono(methyl ether) (14.2 g, 100%) as a golden oil. 1 HNMR (400 MHz, CDC13, δ) : 7.34 (m, 4H), 7.22 (m, 6H), 4.38 (m, 2H), 3.73 (m, 2H),
3.64 (m, 24H), 3.54 (m, 2H), 3.38 (s, 3H).
[0108] Platinium I
oxide hydrate (200 mg) was added to a solution of the mono
phosphate diphenyl ester of PEG-350 mono(methyl ether) prepared above (14.2 g,
24.4 mmol) dissolved in acetic acid, and the resulting suspension was heated to 50 °C
and placed under an atmosphere of H2 until hydrogen uptake ceased. The reaction was
filtered through a celite pad to remove catalyst and the solvent was removed in vacuo
to leave the desired product mono phosphate of PEG-350 mono(methyl ether) (10.49
g, 100%) as a clear, yellow oil. 1 H NMR (400 MHz, CDC13, δ) : 4.20 (m, 2H), 3.67
(m, 24 H), 3.56 (m, 2H), 3.39 (s, 3H).
[0109] To a colloidal suspension of superparamagnetic iron oxide nanoparticles
(SPIO cores solution in benzyl alcohol) diluted to 1 mg Fe/mL with THF was added
the mono phosphate of PEG-350 mono(methyl ether) (2 mol of phosphate groups per
mol of Fe) and the resulting suspension was heated at 50 °C for 16 h . The reaction
was then cooled to room temperature, diluted with water, and the brown aqueous
solution was washed three times with hexanes. Any remaining volatiles in the
aqueous layer were removed in vacuo and the resulting nanoparticles were purified by
washing with H20 against a 30 kDa molecular cutoff filter using tangential flow
filtration to provide a suspension of nanoparticle composition XVII. The particles
had a hydrodynamic diameter of 50 nm, as measured by dynamic light scattering.
After 1 week of storage in water the particles had a hydrodynamic diameter greater
than 100 nm.
XVI I
Comparative Example 5 : Comparison of stability of L2-bisphosphate, 1,3-
bisphosphate, and monophosphate coated SPIOs[0110] Data are gathered in the Table below which compare the properties of
nanoparticle compositions provided by the present invention with a nanoparticle
composition not comprising a phosphorylated polyol comprising at least two
phosphate groups, the PEG-350 phosphate. The effect of a "second" phosphate group
is striking in that it renders the nanoparticle composition both more stable in terms if
change in hydrodynamic diameter (DH) as determined by dynamic light scattering.
Table: Stability and Zeta Potential of Coated SPIOs
Nanoparticle Coating DH post DH 2 weeks post Zeta
synthesis synthesis Potential
PEG-350 Phosphate* 50 ± 1 nm > 100 nm 7.3 mV
1,2-BPP350 SPIO† 9 ± 1 nm 9 ± 1 nm -5.0 mV
1,3-BPP350 SPIO† 9.5 ± 1 nm 8.4 ± 1 nm -1.7 mV
* mono phosphate (also referred to herein as the mono phosphate of PEG-350
mono(methyl ether), † bisphosphate
[0111] Additional data are gathered in the following Table which further illustrate the
advantages of the nanoparticle compositions provided by the present invention. The
data highlight the importance of having at least two phosphate groups present in the
phosphorylated polyol used to stabilize the nanoparticulate metal oxide (here
nanoparticulate superparamagnetic iron oxide, referred to simply as SPIO or SPIOs)
and the advantages provided by stabilizers comprising two phosphate groups and one
or more hydrophilic groups of the polyalkylene ether type, for example polyethylene
ether groups derived from the mono methyl ether of PEG350 or the mono methyl
ether of PEG2000. Significantly, at least some of the nanoparticle compositions
provided by the present invention are stable under autoclaving conditions, which
characteristic may serve as a threshold indicator of suitability for a material's use in
human medical imaging techniques. It is emphasized that the data presented are for
nanoparticle compositions comprising the indicated stabilizer compounds as opposed
to the stabilizer compounds themselves in the absence of the nanoparticulate metal
oxide.
Table: Stability of Coated SPIOs As A Function Of Stabilizer Structure1,2-Bisphosphate Stabilizer Monophosphate Stabilizer
1,2-BPP350 PEG3 50-monophosphate
• DH = 9 nm • DH = >50 nm
• Stable in 150 mM saline • Unstable in 150 mM saline
solution, 2 days at 40°C solution, 2 days at 40°C
• TFF stable • Unstable to TFF
1,2-BPP2000 PEG2000-monophosphate
• DH = 16 nm • DH = 22 nm
• Stable in 150 mM saline • Stable in 150 mM saline solution, 2
solution, 2 days at 40°C days at 40°C
• TFF stable • TFF stable
• Autoclave stable, 121°C 15 min • Unstable to autoclave, 12 1°C 15
min
^Tangential Flow Filtration (purification)
Example 16: In vivo imaging of tumors by RI
[0001] All procedures involving animals were completed under protocols
approved by the GE Global Research Institutional Animal Care and Use Committee.
Tumors were induced in female Fischer 344 rats (-150 g) by subcutaneous injection
of 2xl0 6 Mat B III cells (ATCC# CRL1666, ATCC, Manassas, VA) in 0.1 mL
Hank's balanced saline solution. The injection site was located dorsally between the
shoulder blades. The tumors were imaged 12 days after implantation, when the
tumors were - 1 cm in diameter.
[0002] Imaging was conducted on a clinical 3 Tesla GE MR750 scanner using
a custom-built, - 6 cm solenoid receiver RF coil. To prepare for imaging, the rats
were anesthetized by IP injection of ketamine and diazepam using 55 and 3.8 mg/kg
doses, respectively. Once immobile, a saline primed 1 F tail vein catheter (MTV-02,
Strategic Applications Inc., Libertyville, IL) was placed in a lateral tail vein and
secured with tape. The prepared animal was then placed within the RF coil and
positioned within the bore of the scanner. A pre-injection image set was acquired,
and then, without moving the table or the animal, the 1-2 bisphosphate-PEG (Mw = 2
kDa) coated superparamagnetic iron oxide nanoparticles were injected via the catheter
by a saline flush (-0.4 mL). Following injection, image sets were collectedthroughout a dynamic acquisition period of -30 minutes. For the injection, the
nanoparticle composition (SPIO agent) was formulated in 5% aqueous mannitol at a
concentration of 2 mg Fe/mL and was dosed at 2 mg Fe/kg body weight.
[0003] A 3D fast gradient echo pulse sequence was employed that allowed
simultaneous collection of images at 10 echo times. The imaging slab was positioned
via the graphical prescription interface such that the tumor was centered within the
transaxial slices and the coverage included the majority of the tumor in depth. The
pulse sequence parameters were as follows: pulse sequence: 3D ME fGRE; TE:
ranged from 4.0 to 65.4 ms, with 6.8 ms spacing; TR: 70.4 ms; flip angle: 25 degrees;
bandwidth: 62.5 MHz; matrix: 256x192; slice thickness: 0.6 mm; field of view: 9 cm,
yielding a voxel size of 0.35x0.35x0.6 mm. The sequence acquisition time was ~2
min.
[0004] The imaging data sets were analyzed using a custom software tool
(CineTool v8.0.2, GE Healthcare) built upon the IDL platform (IDL v. 6.3, ITT Corp.,
Boulder, CO). In brief, the image analysis tool allowed manual drawing of 3D
regions of in interest (ROIs) on the pre-injection series with subsequent calculation of
the T2* time constant and extrapolated intensity at TE = 0 by exponential regression
for every voxel within the drawn ROIs at all time points. These data were used for
estimation of physiologic parameters including tumor blood volume and vascular
permeability. Representative images and difference maps are given in FIG. 2 . The
FIG. 2 illustrates the representative Tl-weighted images (TE = 4.0 ms) before
injection of the iron oxide nanoparticle composition (A) and 30 min following
injection of the iron oxide nanoparticle composition (B). The tumor region (arrow)
shows more enhancement than muscle (arrow head), as demonstrated by signal
intensity difference map (C). T2*-weighted images (TE = 24.5 ms) for the same slice
before administration of iron oxide nanoparticle composition (D) and 15 m following
the administration of iron oxide nanoparticle composition (E). Difference map of the
R2* relaxation rate (F) exhibits differentiation of tumor from muscle tissue.
[0112] While only certain features of the invention have been illustrated and
described herein, many modifications and changes will occur to those skilled in theart. It is, therefore, to be understood that the appended claims are intended to cover
all such modifications and changes as fall within the true spirit of the invention.Claims:
1. A nanoparticle composition, comprising:
a nanoparticulate metal oxide; and
a phosphorylated polyol comprising at least two phosphate groups,
wherein the phosphorylated polyol comprises one or more hydrophilic groups
selected from the group consisting of polyethylene ether moieties, polypropylene
ether moieties, polybutylene ether moieties, and combinations of two or more of the
foregoing hydrophilic moieties.
2 . The composition of claim 1, wherein the phosphorylated polyol
comprises at least one of an ester group, amide group, a carbamate group, a urea
group, or a carbonate group.
3 . The composition of claim 1, wherein the phosphorylated polyol has
structure V
wherein n is an integer from about 16 to about 150 and R is an alkyl group.4 . The composition of claim 1, wherein the phosphorylated polyol has
structure VI
wherein n is an integer from about 16 to about 150 and R1 is an alkyl group.
5 . The composition of claim 1, wherein the phosphorylated polyol
comprising at least two phosphate groups and one or more hydrophilic groups has
structure XVIII
XVI I I
wherein O-R2 is independently at each occurrence a phosphate group, a
hydroxy group, or a polyethylene ether moiety.
6 . The composition of claim 1, wherein the nanoparticulate metal oxide
comprises superparamagnetic iron oxide.
7 . The composition of claim 1, wherein the nanoparticulate metal oxide is
tantalum oxide.8 . A nanoparticle composition of claim 1 comprising:
a nanoparticulate iron oxide core; and
a shell comprising a phosphorylated polyol comprising at least two phosphate
groups, wherein at least two of the phosphate groups occupy positions in the
phosphorylated polyol which constitute a 1,2 or 1,3 spatial relationship to one another
and the polyol comprises a hydrophilic group selected from the group consisting of
polyethylene ether moieties, polypropylene ether moieties, polybutylene ether
moieties, and combinations of two or more of the foregoing hydrophilic moieties.
9 . A nanoparticle composition of claim 1 comprising:
a nanoparticulate metal oxide core, wherein the metal oxide comprises a metal
selected from the group consisting of iron, tantalum, zirconium, and hafnium; and
a shell comprising a phosphorylated polyol comprising at least two phosphate
groups, wherein at least two of the phosphate groups occupy positions in the
phosphorylated polyol which constitute a 1,2 or 1,3 spatial relationship to one another
and the polyol comprises a hydrophilic group selected from the group consisting of
polyethylene ether moieties, polypropylene ether moieties, polybutylene ether
moieties, and combinations of two or more of the foregoing hydrophilic moieties.
10. A nanoparticle composition as claimed in any of claims 8 or 9 further
comprising a pharmaceutically acceptable carrier or excipient.
11. A process for making a nanoparticle composition comprising:contacting a nanoparticulate metal oxide core with a shell composition
comprising a phosphorylated polyol comprising at least two phosphate groups and
one or more hydrophilic groups selected from the group consisting of polyethylene
ether moieties, polypropylene ether moieties, polybutylene ether moieties, and
combinations of two or more of the foregoing hydrophilic moieties.
12. A process of diagnostic imaging comprising:
(a) administering a diagnostic agent composition to a subject, wherein the
diagnostic agent composition comprises a nanoparticle composition comprising a
nanoparticulate metal oxide selected from the group consisting of iron oxide,
manganese oxide, tantalum oxide, zirconium oxide, hafnium oxide, and combinations
of two or more of the foregoing metal oxides; and a phosphorylated polyol
comprising at least two phosphate groups and one or more hydrophilic groups
selected from the group consisting of polyethylene ether moieties, polypropylene
ether moieties, polybutylene ether moieties, and combinations of two or more of the
foregoing hydrophilic moieties; and a pharmaceutically acceptable carrier or
excipient; and
(b) subjecting the subject to diagnostic imaging, wherein the nanoparticle
composition acts as a contrast agent.